EP2492356B1 - Verfahren zum Nachweis von methyliertem DNA in einer Probe - Google Patents

Verfahren zum Nachweis von methyliertem DNA in einer Probe Download PDF

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EP2492356B1
EP2492356B1 EP12157307.5A EP12157307A EP2492356B1 EP 2492356 B1 EP2492356 B1 EP 2492356B1 EP 12157307 A EP12157307 A EP 12157307A EP 2492356 B1 EP2492356 B1 EP 2492356B1
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methylated dna
sample
dna
methylated
binding
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EP2492356A1 (de
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Ayako Sakai
Masahiro Kajita
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Sysmex Corp
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Sysmex Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates to a method of detecting methylated DNA in a sample containing DNA.
  • C cytosine
  • DNA methylation is one of control mechanisms of gene expression and plays an important role in various physiological and pathological phenomena such as early embryonic development, tissue-specific gene expression, genomic imprinting and X chromosome inactivation which are phenomena unique to mammals, chromosome stabilization, and timing of DNA replication. Furthermore, it has been revealed that abnormalities in methylation of DNA, i.e., gene silencing due to the DNA methylation, is involved in diseases such as cancer. Thus, the importance of detecting methylated DNA in various genes has been recently increased.
  • the method of detecting methylated DNA various methods are known in the art. Examples thereof include a bisulfite sequencing method, a method of using a methylation-sensitive restriction enzyme, and a method of using methylated DNA immunoprecipitation (the MeDIP method).
  • the bisulfite sequencing method unmethylated cytosine in DNA is converted to uracil by an effect of bisulfite, the base sequence is determined, and a methylated site in DNA is detected.
  • the method of using a methylation-sensitive restriction enzyme the fact that the methylation-sensitive restriction enzyme cannot cleave the methylated recognition sequence is utilized and methylated DNA is detected by examining the cleavage results.
  • MeDIP immunoprecipitation is performed using an antibody specifically recognizing methylated DNA or a methylated DNA binding protein, the obtained methylated DNA is subjected to microarray analysis, and the methylated DNA is detected. This method is known as the MeDIP-Chip method.
  • JP-A Japanese Patent Application Laid-Open (JP-A) No. 2008-263961 discloses a method of detecting methylated DNA using a methylation-sensitive restriction enzyme comprising: immobilizing methylated DNA in a sample on a solid phase using an anti-methylated DNA antibody; and preforming digestive treatment with a methylation-sensitive restriction enzyme.
  • An objective of the present invention is to provide a novel method of detecting methylated DNA in a sample.
  • the present inventors have surprisingly found that when a protein capable of binding to methylated DNA is bound to methylated DNA, the methylated DNA is not completely degradated by deoxyribonuclease and still remains, and the obtained deoxyribonuclease degradation product does not have an affect in a detection process. Thus, the present invention has been completed.
  • the present invention provides a novel method of detecting methylated DNA in a sample. According to the present invention, it is possible to omit cleaning and purifying processes which are needed to improve detection accuracy in conventional methods of detecting methylated DNA. In this case, methylated DNA in a sample can be more simply detected.
  • CpG site used herein means a site where a cytosine (C) is adjacent to a guanine (G) in the base sequence of DNA in this order in a direction from 5' to 3'.
  • the character of "p" of CpG represents a phosphodiester linkage between cytosine and guanine.
  • methylated CpG site means a CpG site in which the 5th position of cytosine is modified by methylation. That is, at the methylated CpG site, 5-methylcytosine (methylated cytosine) is adjacent to guanine in this order in a direction from 5' to 3'.
  • methylated DNA used herein means DNA containing at least one 5-methylcytosine.
  • a sample which may include methylated DNA is brought into contact with the protein capable of binding to methylated DNA.
  • the methylated DNA can be bound to the above protein because of the contact (hereinafter, the process is called a "binding process").
  • the methylated DNA bound to the above protein is not completely degradated by deoxyribonuclease which is used in the following process. That is, the binding to the above protein allows 5-methylcytosine in methylated DNA and its peripheral region to be saved from degradation by deoxyribonuclease.
  • the sample to be subjected to the detection method of the present invention is not particularly limited as long as it is the sample which may include methylated DNA.
  • the sample include a DNA containing sample prepared from a biological sample and a sample containing a synthetic polynucleotide which contains at least one 5-methylcytosine.
  • the biological sample include cultured cells or blood, body fluids, tissues and cells collected from organisms.
  • the "blood” used herein may be any of whole blood and serum and blood plasma obtained from the whole blood.
  • tissues and “cells” used herein include cultures of the tissues and cells collected from organisms.
  • the sample which may include methylated DNA is a sample prepared from blood, body fluids, tissues or cells collected from organisms
  • the results of the detection method of the present invention can be used for the diagnosis and determination of diseases associated with methylated DNA.
  • preparation of a DNA containing sample from a biological sample can be performed by a known method in the art.
  • a sample can be prepared by the steps including mixing a solubilized liquid which contains a surfactant for solubilizing cells or tissues (sodium cholate, sodium dodecyl sulfate, etc.) with a biological sample, subjecting the mixture to physical processes (stirring, homogenization, ultrasonic fragmentation, etc.), releasing DNA included in the biological sample into a solubilized liquid, and extracting the DNA.
  • the extracted DNA may be purified by a known method in the art.
  • the extraction and purification of DNA from the biological sample can be performed using a commercially available kit.
  • DNA in a sample is preferably a DNA fragment with 100 to 1000 bp.
  • the methylated DNA can be efficiently bound to the protein capable of binding to methylated DNA by fragmenting the DNA included in the sample to the length.
  • DNA fragmentation can be performed by a known method in the art, such as a physical process, a chemical process or a restriction enzyme process.
  • a physical process for example, ultrasonic fragmentation is cited.
  • a chemical process for example, alkaline treatment using sodium hydroxide is cited.
  • the restriction enzyme process the restriction enzyme can be appropriately selected based on a base sequence of a target DNA. For example, MseI, BamHI, and the like can be used.
  • it is preferable that DNA in a sample is fragmented by the physical process.
  • methylated DNA which may be included in a sample may be single-stranded methylated DNA. That is, the above sample is a sample which may include single-stranded methylated DNA.
  • a process of detecting methylated DNA to be described later is a process of detecting single-stranded methylated DNA is included in the scope of the present invention.
  • a process of modifying the DNA included in the sample to a single-strand may be performed before the binding process. The process is known in the art.
  • the DNA in the sample can be modified to a single-strand by heating the sample which may include methylated DNA at about 95°C and immediately cooling it to 4°C.
  • the protein capable of binding to methylated DNA is not particularly limited as long as it is a protein capable of recognizing and binding to 5-methylcytosine in DNA or the methylated CpG site.
  • the protein include an anti-methylated DNA antibody and a methylated DNA binding protein.
  • the anti-methylated DNA antibody is preferred. Particularly, when single-stranded methylated DNA is detected, it is desirable to use the anti-methylated DNA antibody.
  • the anti-methylated DNA antibody examples include an anti-methylated cytosine antibody and an anti-methylated CpG antibody.
  • an anti-methylated cytidine antibody can be used as the anti-methylated DNA antibody.
  • the anti-methylated DNA antibody may be either a polyclonal antibody or a monoclonal antibody.
  • an active fragment which is obtained by fragmenting the anti-methylated DNA antibody may be used.
  • the active fragment is not particularly limited as long as it is a fragment which does not lose a specific binding activity to methylated DNA. Examples thereof include a Fab fragment, a F(ab') 2 fragment, and a sFv fragment.
  • the active fragment can be prepared, for example, by cleaving a purified anti-methylated DNA monoclonal antibody by an enzyme such as papain, pepsin or trypsin.
  • a commercially available anti-methylated DNA antibody may be used.
  • Examples of the commercially available anti-methylated DNA antibody include antibodies shown in Table 1.
  • an anti-methylated DNA antibody produced by a known method in the art may be used.
  • the anti-methylated DNA antibody can be produced, for example, by the following procedure. First, an appropriate mammal (e.g. rats and mice) is immunized with methylated DNA such as 5-methylcytosine as an immunogen together with an adjuvant, if desired. Then, an antibody-producing cell that produces an antibody against an immunogen is selected by screening it from spleen cells of the immunized animal. The obtained antibody-producing cell is fused with a myeloma cell to produce a hybridoma.
  • methylated DNA such as 5-methylcytosine as an immunogen together with an adjuvant, if desired.
  • an antibody-producing cell that produces an antibody against an immunogen is selected by screening it from spleen cells of the immunized animal.
  • the obtained antibody-producing cell is fused with a myeloma cell to produce a hybridoma.
  • the hybridoma is screened and thus a hybridoma that produces an antibody having a specific binding activity to methylated DNA is obtained.
  • the anti-methylated DNA antibody can be obtained from the ascitic fluid obtained by administering the culture supernatant of the obtained hybridoma or the hybridoma to the abdominal cavity of a mouse.
  • hybridoma SCR2 deposited with The National Institute of Technology and Evaluation (2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan.) under Accession No. NITE BP-805 on August 25, 2009; and hybridomas SCR1, SCR3, and SCR6 deposited under Accession Nos. NITE BP-810, NITE BP-811, and NITE BP-812, respectively on September 10, 2009.
  • anti-methylated DNA antibodies which are produced from the hybridomas SCR1, SCR2, SCR3, and SCR6 can be used.
  • a methylated DNA binding protein examples include MBD1 (methyl cytosine binding domainprotein 1), MBD2 (methyl cytosine binding domain protein 2), MBD4 (methyl cytosine binding domain protein 4), and MeCP2(methyl CpG binding protein 2). These proteins in themselves are known in the art.
  • the methylated DNA binding protein may be a variant having deletion, substitution, or addition of one or more amino acids in an amino acid sequence of a wild type as long as it can specifically recognize and bind to methylated DNA. A method of producing such a variant itself is known in the art.
  • the contact of the sample which may include methylated DNA with the protein capable of binding to methylated DNA can be performed by adding the protein to a sample.
  • the additive amount of the protein capable of binding to methylated DNA is not particularly limited and it may be an additive amount capable of ensuring the amount of methylated DNA which can be sufficiently detected in the following detection process.
  • the additive amount of the anti-methylated DNA antibody may be from about 1 ng to 10 ⁇ g.
  • the contact conditions (ambient temperature and time) vary depending on the type of the protein capable of binding to methylated DNA and are usually as follows: at 4 to 42°C for about 10 minutes to 24 hours.
  • the sample obtained in the binding process is brought into contact with at least one deoxyribonuclease to degrade DNA in the sample (hereafter the process is called a "degradation process").
  • a degradation process DNA which is not bound to the protein capable of binding to methylated DNA (i.e., unmethylated DNA) is degradated by deoxyribonuclease.
  • unmethylated DNA is degradated by the binding.
  • unmethylated DNA can be removed by the binding process and the degradation process.
  • deoxyribonuclease may be either of endonuclease or exonuclease.
  • a restriction enzyme may be used as deoxyribonuclease.
  • a DNA cleavage site is limited to a recognition region of the restriction enzyme. Therefore, when the sample obtained by the degradation process is directly used for the subsequent detection process, it should be noted that degradated products have no affect on the following detection process. For example, unmethylated DNA can be sufficiently degradated using a plurality of restriction enzymes.
  • deoxyribonuclease is at least one selected from the group consisting of deoxyribonuclease I (DNaseI), exonuclease I, lambda exonuclease, T7 exonuclease, exonuclease III, RecJ exonuclease, exonuclease T, BAL31 nuclease, Mung Bean nuclease, the Micrococcaceae nuclease, and T7 endonuclease.
  • DNaseI deoxyribonuclease I
  • exonuclease I lambda exonuclease
  • T7 exonuclease exonuclease III
  • RecJ exonuclease exonuclease T
  • BAL31 nuclease BAL31 nuclease
  • Mung Bean nuclease Mung Bean nuclease
  • T7 endonuclease
  • At least one deoxyribonuclease to be used is a deoxyribonuclease different from a methylation-sensitive restriction enzyme capable of degradating single-stranded DNA.
  • methylation-sensitive restriction enzyme capable of degradating a single-stranded DNA means a restriction enzyme that does not cleave a recognition sequence containing methylated cytosine in a single-stranded DNA and can cleave only a recognition sequence containing unmethylated cytosine.
  • a restriction enzyme for example, HhaI is cited.
  • at least one methylation insensitive deoxyribonuclease is used.
  • the contact of the sample obtained in the binding process with deoxyribonuclease can be performed by adding deoxyribonuclease to a sample.
  • the additive amount of deoxyribonuclease is not particularly limited and it may be an additive amount capable of sufficiently degrading unmethylated DNA in a subsequent detection process.
  • the additive amount of DNaseI may be from about 0.1 to 120 U.
  • the contact conditions vary depending on the type of deoxyribonuclease and are usually as follows: at 4 to 42°C for about 10 minutes to 24 hours.
  • the sample obtained by the degradation process can be directly used in the following detection process.
  • the process of cleaning to remove unmethylated DNA and the process of purifying methylated DNA can be omitted.
  • the methods of the invention do not comprise a step of separating methylated from unmethylated DNA prior to detection of methylated DNA.
  • the step of detecting methylated DNA which is not degraded by the deoxyribonuclease (due to the binding of the sample obtained by the degradation process to the protein) follows directly after the step of bringing the sample (obtained in the binding process) into contact with at least one deoxyribonuclease to degrade DNA in the sample.
  • a nucleic acid amplification method may be executed using methylated DNA included in the sample obtained by the degradation process as a template.
  • the DNA amplification method itself is not particularly limited as long as it is a known DNA amplification method in the art. Examples thereof include in vitro transcription (IVT) amplification, SPIA (trademark) amplification, and GenomiPhi amplification.
  • methylated DNA which is not degraded by deoxyribonuclease is detected by the binding of the sample obtained by the degradation process with the protein capable of binding to methylated DNA (hereafter, the process is called as a "detection process").
  • frequency of methylation used herein means the number or ratio of the methylated CpG site of all CpG sites or arbitrary CpG sites present in a target region in methylated DNA.
  • the detection of methylated DNA is not particularly limited as long as it is a method which can detect the methylated DNA which is not degraded by deoxyribonuclease in the degradation process, such as determination of methylation of a target DNA, determination of a base sequence of methylated DNA, and comprehensive analysis of methylated DNA.
  • the method to be used in the detection process is not particularly limited as long as it can detect the methylated DNA which is not degraded by deoxyribonuclease in the degradation process and it can be appropriately selected from known methods in the art.
  • the method include a nucleic acid amplification method, a nucleotide sequencing method, and a microarray method.
  • the PCR assay is performed using a primer set which is designed so that a CpG site in a target DNA is sandwiched and the presence of methylation of a target DNA is determined from the presence of amplified products.
  • the content of methylated DNA in the sample can also be quantified by the quantitative PCR method.
  • a high-speed sequencing method (a next-generation sequencing method), which is one of the nucleotide sequencing methods, is used in the detection process, methylated DNA can be comprehensively analyzed. Examples of a sequencer to be used for the high-speed sequencing method include ABI3730x1 (Life Technologies) and GS FLX Titanium (Roche).
  • the microarray method can also be used in the detection process.
  • a microarray is not particularly limited and a DNA microarray or a DNA chip is preferred.
  • a commercially available microarray such as GeneChip (registered trademark, manufactured by Affymetrix) or a microarray produced by a known method in the art may be used. If a tiling array which is a microarray in which probes having a base sequence extracted at equal intervals from a whole genome region or a specific region are disposed in a tile shape is used in the detection process, methylated DNA can be comprehensively analyzed.
  • the DNA in the sample is preferably labeled with a labeling substance known in the art. Therefore, the detection method of the present invention may further include a process of labeling DNA in a sample.
  • the labeling substance include fluorescent substances, haptens such as biotin, and radioactive substances.
  • the fluorescent substances include Cy3, Cy5, Alexa Fluor (trademark), and FITC.
  • the signal is easily measured by labeling DNA. Methods for labeling the DNA with the labeling substance are known in the art.
  • the signals may be signals suitable for the type of the microarray.
  • the signals may be electric signals which are generated in the presence of DNA hybridizing to each probe of the microarray, or when the DNA in the sample is labeled as described above, the signals may be fluorescence signals, luminescence signals or radioactive signals.
  • Each of the signals may be detected using a scanner installed in a general microarray analyzer.
  • the scanner may be GeneChip (registered trademark), Scanner 3000 7G (Affymetrix, Inc.) or the like.
  • 6MeCG oligonucleotide i.e., oligonucleotide containing six of 5-methylcytosine was used.
  • the base sequence of 6MeCG oligonucleotide is shown below. ("m5c" in the base sequence represents 5-methylcytosine.))
  • a 10 pM concentration of an aqueous solution of 6MeCG oligonucleotide was prepared as a 6MeCG oligonucleotide solution.
  • the 6MeCG oligonucleotide solution was heated at 95°C for 10 minutes and the modified solution was allowed to stand on ice for 1 minute. 2 ⁇ l was taken from the modified 6MeCG oligonucleotide solution and this was used as an input sample. The remaining solution was used as a sample to be subjected to the detection method of the present invention.
  • the sample was divided into two. An anti-methylated DNA antibody (1 ⁇ g) was added to one of them to prepare a specimen sample (100 ⁇ l) and the anti-methylated DNA antibody was not added to the other one to prepare a control sample (100 ⁇ l). Each sample was incubated at 25 °C for 2 hours.
  • the anti-methylated DNA antibody used in this example is a monoclonal antibody obtained from hybridoma SCR2 deposited with The National Institute of Technology and Evaluation (2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan.) under Accession No. NITE BP-805 on August 25, 2009.
  • DNaseI i.e., a type of deoxyribonuclease (2 U/ ⁇ l:NEB) was added to the specimen sample and the control sample and each mixture was reacted at 37°C for 1 hour. After the reaction, each sample was heated at 75°C for 10 minutes to deactivate DNaseI.
  • a primer set for detecting 6MeCG oligonucleotide a primer set represented by SEQ ID NO: 2 and SEQ ID NO: 3 was used.
  • the base sequences of the primer set are shown below.
  • the obtained reaction solution was subjected to electrophoresis using 3% agarose gel and then the presence of amplified products was confirmed.
  • the results are shown in FIG. 1 . From Fig. 1 , it is found that bands of amplified products were not detected in the sample to which the anti-methylated DNA antibody was not added, while the bands were detected in the sample to which the antibody was added. This suggested that the degradation of the oligonucleotide by DNaseI was inhibited by allowing the anti-methylated DNA antibody to be bound to 6MeCG oligonucleotide, and thus 6MeCG oligonucleotide could be detected.
  • a solution of 6MeCG oligonucleotide was prepared in the same manner as described in Example 1, and an input sample and a sample being subjected to the detection method of the present invention were obtained from the solution.
  • Exonuclease I 20 U/ ⁇ l:NEB
  • a type of deoxyribonuclease was added to each of the specimen and control samples and each mixture was reacted at 37°C for 1 hour. After the reaction, each sample was heated at 80°C for 20 minutes to deactivate Exonuclease I.
  • the obtained reaction solution was subjected to electrophoresis using 3% agarose gel and then the presence of amplified products was confirmed. The results are shown in Fig. 2 .
  • a promoter region of GSTP1 gene which was known to be frequently modified by methylation in genomic DNA of the breast cancer cell line MCF7 was selected.
  • the base sequence in the promoter region of GSTP1 gene is known in the art.
  • CGF-1 region a region which was present in the 14th human chromosome and was not modified by methylation because of having no CpG site.
  • the base sequence of the CGF-1 region is shown below.
  • a solution of genomic DNA extracted from MCF7 cells was fragmented by ultrasonic fragmentation using Bio-raptor (registered trademark, manufactured by COSMO BIO Co., Ltd.) to prepare a solution of genomic DNA fragments with 500 bp or less.
  • the prepared solution of genomic DNA fragments was modified by heating at 95°C for 10 minutes and the resultant solution was allowed to stand on ice for 1 minute to obtain a solution of single stranded DNA fragments.
  • An aliquot was taken from a sample prepared by adjusting the obtained solution of single stranded DNA fragments so that the amount of DNA was 2 ng/ ⁇ l and the aliquot was used as an input sample.
  • the anti-methylated DNA antibody (1 ⁇ g) obtained from hybridoma SCR2 was added to50 ⁇ l of the sample and the mixture was used as a specimen sample (100 ⁇ l). Pure water was added to 50 ⁇ l of the sample and the mixture was used as a control sample (100 ⁇ l). Each sample was incubated at 25 °C for 2 hours.
  • the specimen sample was divided into two. 0.5 ⁇ l of DNaseI (2 U/ ⁇ l:NEB) was added one of them and 1 ⁇ l of DNaseI was added to the other one.
  • the control sample was divided into two. 0.5 ⁇ l of DNaseI (2 U/ ⁇ l:NEB) was added one of them and 1 ⁇ l of DNaseI was added to the other one.
  • the quantitative PCR method was performed. In order to confirm that methylated DNA is specifically detected by the detection method of the present invention, degradation of unmethylated DNA in a sample was examined by the quantitative PCR method.
  • a primer set for amplifying a promoter region of GSTP1 gene was used as a primer set for detecting methylated DNA.
  • the base sequences of the primer set are shown below. F:5'- GAGGCCTTCGCTGGAGTT -3'(SEQ ID NO 5)
  • a primer set for amplifying a CGF-1 region was used as a primer set for detecting unmethylated DNA.
  • the base sequences of the primer set are shown below.
  • the obtained reaction solution was subjected to electrophoresis using 3% agarose gel and then the presence of amplified products was confirmed.
  • the results are shown in Fig. 3 . From Fig. 3 , it is found that bands of amplified products in the GSTP1 promoter region were not detected in the sample to which the anti-methylated DNA antibody was not added, while the bands were detected in the sample to which the antibody was added. On the other hand, it is found that bands of amplified products in the CGF-1 region were not detected regardless of whether the addition of the anti-methylated DNA antibody was carried out.
  • a solution containing a single-stranded genomic DNA derived from MCF7 was prepared in the same manner as described in Example 3 and an input sample and a sample being subjected to the detection method of the present invention were obtained from the solution.
  • Exonuclease I 20 U/ ⁇ l:NEB
  • a PCR reaction solution was prepared in the same manner as described in Example 3 and the quantitative PCR method was performed.
  • the reaction conditions of quantitative PCR are as follows.
  • the obtained reaction solution was subjected to electrophoresis using 3% agarose gel and then the presence of amplified products was confirmed.
  • the results are shown in Fig. 4 . From Fig. 4 , it is found that bands of amplified products in the GSTP1 promoter region were not detected in the sample to which the anti-methylated DNA antibody was not added, while the bands were detected in the sample to which the antibody was added. On the other hand, it is found that bands of amplified products in the CGF-1 region were not detected regardless of whether the addition of the anti-methylated DNA antibody was carried out.

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Claims (8)

  1. Verfahren zum Nachweis methylierter DNA in einer Probe, welches Folgendes umfasst:
    in Kontakt bringen einer Probe, welche möglicherweise methylierte DNA enthält, mit einem Protein, welches in der Lage ist, an methylierte DNA zu binden, zum Binden der methylierten DNA in der Probe an das Protein;
    in Kontakt bringen der Probe, welche in dem Bindungsprozess erhalten wurde, mit mindestens einer Deoxyribonuklease zum Zersetzen von DNA in der Probe; und
    Nachweisen methylierter DNA, welche nicht durch die Deoxyribonuklease zersetzt wird, durch die Bindung der Probe, welche durch den Zersetzungsprozess erhalten wurde, an das Protein;
    wobei die mindestens eine Deoxyribonuklease eine andere Deoxyribonuklease als ein methylierungssensitives Restriktionsenzym, welches in der Lage ist, Einzelstrang-DNA zu zersetzen, ist.
  2. Verfahren nach Anspruch 1, wobei ein Protein, welches zum Binden an die methylierte DNA in der Lage ist, ein Antikörper gegen methylierte DNA oder ein Bindungsprotein für methylierte DNA ist.
  3. Verfahren nach Anspruch 1, wobei das Protein, welches zum Binden an die methylierte DNA in der Lage ist, ein Antikörper gegen methylierte DNA ist.
  4. Verfahren nach Anspruch 1, wobei das Protein, welches zum Binden an die methylierte DNA in der Lage ist, mindestens ein Bindungsprotein für methylierte DNA ausgewählt aus der Gruppe bestehend aus MBD1, MBD2, MBD4 und MeCP2 ist.
  5. Verfahren nach einem der Ansprüche 1 bis 4, wobei die Probe kultivierte Zellen oder eine Probe hergestellt aus Blut, Körperfluiden, Geweben oder Zellen entnommen aus Organismen sind/ist.
  6. Verfahren nach einem der Ansprüche 1 bis 5, wobei die Deoxyribonuklease mindestens eine ausgewählt aus der Gruppe bestehend aus Deoxyribonuklease I, Exonuklease I, Lambda-Exonuklease, T7-Exonuklease, Exonuklease III, RecJ-Exonuklease, Exonuklease T, BAL31-Nuklease, Mungobohnen-Nuklease, der Mikrokokken-Nuklease und T7-Endonuklease ist.
  7. Verfahren nach einem der Ansprüche 1 bis 6, wobei die Probe eine Probe ist, welche methylierte Einzelstrang-DNA enthalten kann, und die methylierte Einzelstrang-DNA in dem Prozess des Nachweises methylierter DNA nachgewiesen wird.
  8. Verfahren nach einem der Ansprüche 1 bis 7, wobei methylierte DNA mittels eines Nukleinsäure-Amplifikationsverfahrens, eines Nukleotidsequenzierungsverfahrens oder eines Mikroarrayverfahrens im Prozess des Nachweises methylierter DNA nachgewiesen wird.
EP12157307.5A 2011-02-28 2012-02-28 Verfahren zum Nachweis von methyliertem DNA in einer Probe Active EP2492356B1 (de)

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EP2492356A1 (de) 2012-08-29
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